The habenula and thalamus arise from two closely related progenitor domains during embryogenesis and they play crucial roles in modulating the forebrain circuitry in a mature brain. The developmental mechanisms underlying the distinct thalamic and habenular connectivity, and the topographic projection of thalamic axons to specific cortical areas are still poorly understood at both transcriptional and axon guidance levels. Our long-term goal is to determine the molecular mechanisms that regulate thalamic and habenular identity and connectivity, and ultimately understand the brain disorders resulting from abnormal formation and/or function of the thalamus and habenula. The overall objective of this application is to determine the molecular control of the guidance decisions during the initial outgrowth of thalamic axons and their subsequent navigation to the cortex. Our central hypothesis is that Gbx2 controls the intrinsic responsiveness of thalamic axons to guidance cues encountered in their path to the cortex by regulating the modification of heparan sulfate and the expression of guidance receptors Robo1 and Robo2. Therefore, our proposed study will provide new insight into the important but less understood role of heparan sulfate in regulating axonal guidance. Furthermore, the elucidation of how Gbx2 controls the intrinsic responsiveness of thalamic axons will enhance our understanding on the establishment of the thalamic connectivity, including the topography of thalamocortical axons. The hypothesis has been formulated on the basis of results from our preliminary studies, including identifications of several downstream targets that may mediate Gbx2 function. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Determine the role of modifications of heparan sulfate in the developing thalamus; and 2) Determine the molecular mechanism that regulates the topography of thalamocortical projections. We will combine in vitro studies (brain slice and explant culture, molecular biology, and biochemistry) and mouse genetics (chimeric, genetic mosaic, inducible genetic fate mapping and conditional knock-out). The approach is innovative, because it employs state-of-the-art mouse genetics, and utilizes different fluorescence proteins to identify all thalamic axons and those with Gbx2 deletion to study the topography of thalamocortical projections. The proposed studies are expected to significantly enhance our understanding on the transcriptional regulation and axonal guidance signaling in the establishment of thalamic projections to the cortex. Ultimately, such knowledge will contribute to our understanding on the pathogenesis or susceptibility of neurological illnesses, such as epilepsy, schizophrenia, bipolar disorder, and autism. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because abnormal formation and function of the habenula and thalamus have been linked to neurological illnesses, such as epilepsy, schizophrenia, bipolar disorder, sleep disorders, and autism. Elucidation of the molecular mechanisms that govern the generation of thalamic and habenular connectivity will be ultimately expected to augment our understanding of the pathogenesis or conditions predisposing to the aforementioned brain disorders.